This invention generally relates to flash spectrofluorimetry, in which a sample under analysis is subjected to flashes of exciting radiation of very short duration compared with a dwell time allowed between flashes and the fluorescence emitted by the sample is measured by a photodetector in order to derive a fluorescence measuring signal characteristic of the sample; in particular, the invention relates to method of and apparatus for flash spectrofluorimetry wherein the fluorescence measuring signal is substantially freed from the vitiating effect thereon of any spurious currents, e.g. dark current, flowing through the photodetector, in particular currents occurring simultaneously with the said signal. Although spurious currents may arise in different ways--as will be later indicated--their net effect is the setting up in the photodetector of a "standing current" that masks the true value of the fluorescence measuring signal, whether said signal is evaluated as a current or a voltage output.
A spark source, or alternatively a continuously energized source cooperating with a light chopper, may of course be used in flash spectrofluorimetry, but in the present state of the art the best balance of operational and constructional advantages resulting from intermittent irradiation is achieved by the use of a plasma discharge source supplied with energization pulses which are (a) of very short duration (measured in microseconds) and sufficiently high peak power (measured in kilowatts) to raise the plasma produced on discharge to the colour temperature required for a continuum of light to be emitted, and (b) of sufficiently low repetition rate (dwell between pulses measured in milliseconds) to ensure that the average power dissipated by the source is maintained within reasonable bounds (typically within tens of watts) so that the life of the source is not unduly curtailed. One notable operational advantage resulting is the ease with which weakly fluorescing samples may be subjected to high level of excitation without any risk of damaging them. Constructionally, the comparatively small power supply required is a significant bonus, but more important, of course, is the closer approximation to the ideal source geometry that can be realized by the compactness of design permitted by the low average power dissipation.
It will be stressed later that the plasma discharge source is the preferred source in the context of the present invention since, in addition to the advantages referred to, it actually facilitates the realization of the present invention by enabling the fluorescence measuring signal to be evaluated over a very short interval of a few tens of microseconds.
An early example of flash spectrofluorimetry in which pulsed high-intensity irradiation of a sample is provided by a plasma discharge source is described in U.S. Pat. No. 3,787,695. In that example the fluorescence of the sample substantially coincident with the duration of each flash is analyzed by scanning it with a continuous interference filter, at a slow enough rate of displacement relative to the flashing frequency to include a conveniently large number of flashes in one complete scan travel, the scan output being detected with a photomultiplier and finally integrated for presentation on a chart recorder. To attenuate the effect of light source fluctuation on the recorded output, a reference photomultiplier is used in addition to the sample photomultiplier and their respective outputs are ratioed. This scheme works well but cannot combat the spurious responses introduced by unavoidable out-of-balances between the two outputs in terms of dark current characteristics, stray light, etc.
In the prior art specification referred to, the flash duration is contained within a few microseconds and the dwell between flashes is some 20 milliseconds. This means that the signal content of each photomultiplier output is of a very short duration compared with the spurious content due to the standing current flowing between successive flashes. In other words, the dark current contribution arising during the dwell period is a major cause of the out-of-balances.
In U.S. Pat. No. 4,049,970, assigned to the present applicant, the problem caused by the out-of-balances was greatly minimized by gating the photomultipliers so that they were only active for the signal duration (i.e. for the duration of the actual fluorescence measuring signal), the effect of any standing current flowing simultaneously with the signal being regarded as too small to be troublesome. The art has now progressed to the point where the effect can no longer be regarded as insignificant in terms of the fidelity of the fluorescence signal measurement, bearing in mind that dark current is not the only spurious factor to be accounted for, other significant factors such as stray light, sample phosphorescence (in measuring fluorescence any contribution to the photodetector output due to phosphorescence of the sample is spurious, of course) and so forth being also present.